RNA ligase is abundant in T4-infected cells and has been purified in high yields. Bacteriophage T4 RNA ligase catalyzes the ATP-dependent ligation of a 5′-phosphoryl-terminated nucleic acid donor (i.e., RNA or DNA) to a 3′-hydroxyl-terminated nucleic acid acceptor. The reaction can be either intramolecular or intermolecular, i.e., the enzyme catalyzes the formation of circular DNA/RNA, linear DNA/RNA dimers, and RNA-DNA or DNA-RNA block co-polymers. The use of a 5′-phosphate, 3′-hydroxyl terminated acceptor and a 5′-phosphate, 3′-phosphate terminated donor limits the reaction to a unique product. Thus, RNA ligase can be an important tool in the synthesis of DNA of defined sequence (McCoy & Gumport, Biochemistry 19:635-642 (1980); Sugino, A. et al., J. Biol. Chem. 252:1732-1738 (1977)).
The practical use of T4 RNA ligase has been demonstrated in many ways. Various ligation-anchored PCR amplification methods have been developed, where an anchor of defined sequence is directly ligated to single strand DNA (following primer extension, e.g., first strand cDNA). The PCR resultant product is amplified using primers specific for both the DNA of interest and the anchor (Apte, A. N., and P. D. Siebert, BioTechniques, 15:890-893 (1993); Troutt, A. B., et al., Proc. Natl. Acad. Sci. USA, 89:9823-9825 (1992); Zhang, X. H., and V. L. Chiang, Nucleic Acids Res., 24:990-991(1996)). Furthermore, T4 RNA ligase has been used in fluorescence-, isotope- or biotin-labeling of the 5′-end of single stranded DNA/RNA molecules (Kinoshita Y., et al., NucleiAcid Res., 25: 3747-3748 (1997)), synthesis of circular hammer head ribozymes (Wang, L., and D. E. Ruffner. Nucleic Acids Res., 26:2502-2504 (1998)), synthesis of dinucleoside polyphosphates (Atencia, E. A., et al., Eur. J. Biochem., 261: 802-811 (1999)), and for the production of composite primers (Kaluz, S., et al., BioTechniques, 19:182-186 (1995)).
RNA ligase activity was originally identified as activity induced through infection of E. coli by T-even bacteriophages (Silber, R et al., Proc. Natl. Acad. USA, 69: 3009-3013 (1972)). The RNA ligase from bacteriophage T4 is the product of gene 63 (Snopek, T. J., et al., Proc. Natl. Acad. Sci. USA, 74:3355-3359 (1977)) and is the best characterized RNA ligase of very few known homologous RNA ligases.
The properties of RNA ligase from bacteriophage T4 have been extensively studied including its ability to catalyze reactions with various substrates (for review see Gumport and Uhlenbeck, in “Gene Amplification and Analysis,” Vol. II: Analysis of Nucleic Acid Structure by Enzymatic Methods, Chirikjian and Papas, eds. Elsevier North Holland, Inc. (1980)). In general, the T4 RNA ligase catalyzes the ATP-dependent formation of a phosphodiester bond between a 3′-hydroxyl nucleic acid acceptor and a 5′-phosphate nucleic acid donor. This includes ligation of two oligonucleotides as well as the circularization of a single oligonucleotide. T4 RNA ligase can use single-stranded nucleic acids as substrates and does not require a complementary template strand to align donor phosphates with acceptor hydroxyls.
5′-phosphorylated oligonucleotides are appropriate donors for the ATP-dependent T4 RNA ligase reaction but the minimal donor is a nucleoside 3′,5′-biphosphate (pNp). The suitable minimal acceptor molecules for the T4 RNA ligase reaction are trinudeoside diphosphates.
T4 RNA ligase is adenylated in the presence of ATP thereby forming a covalent bond between AMP and a lysyl residue. The adenylyl group may then be transferred from the enzyme to the 5′-phosphate of an acceptor nucleic acid. T4 RNA ligase can accept ATP analogues and adenylate nucleic acid substrates with the nucleotide analogue. T4 RNA ligase is able to catalyze a class of reactions that do not require ATP. The enzyme is able to accept a wide variety of ADP derivatives as substrates and join the extra moiety of the ADP derivative to a nucleic acid acceptor with the elimination of AMP. Examples of ADP derivative of this type include ADP-riboflavin and ADP-hexylamine-blotin (see further Gumport and Uhlenbeck, ibid.)
T4 RNA ligase has a greater affinity for RNA than DNA. Although RNA and DNA are equally reactive as donors, DNA is a much less efficient acceptor than RNA. The efficiency of the RNA ligase reaction is also affected by the nucleotide composition of the acceptor with oligo(A) the most efficient acceptor. RNA molecules are also good acceptors for the T4 RNA ligase.
The 5′-phosphate of yeast tRNAPhe is a very poor donor for T4 RNA ligase, indicating that secondary or tertiary structure in the RNA donor molecule is inhibiting the ligase reaction. In contrast, DNA restriction fragments are good donors and little difference is observed between DNA restriction fragments with 5′-staggered ends and blunt ends. On the other hand, the presence of a secondary structure of an RNA acceptor molecule has little effect on the reaction. The 5′-cap (m7 G5′ppp-5′), which is normally formed through addition of methylated guanosine to the 5′ end of eukaryotic mRNA, is neither an acceptor nor a donor for the T4 RNA ligase reaction (Gumport and Uhlenbeck, ibid.).
T4 RNA ligase is a versatile enzyme with new properties continuing to be discovered. For example, T4 RNA ligase has recently been shown to be able catalyze reaction between a 3′-phosphate donor and 5′-hydroxyl acceptor in addition to previously characterized reaction of 5′-phosphate donor and 3′-hydroxyl acceptor (U.S. Pat. No. 6,329,177). T4 RNA ligase has also been shown to have template-mediated DNA ligase activity. Reportedly, the T4 RNA ligase can ligate ends of DNA strands hybridized to RNA, even more efficiently than T4 DNA ligase (U.S. Pat. No. 6,368,801).
Enzymes having RNA ligase activity, but which are apparently not related to the T4 RNA ligase and other homologous proteins in the small family of viral RNA ligases, have been identified. These enzymes may have relatively strict substrate specificity whereas the activity of T4 RNA ligase is the most general RNA joining activity known.
The RNA ligases of T-even bacteriophages apparently belong to a very small family of homologous enzymes. However, it is likely that this is a subfamily of much larger superfamily of ligases including DNA ligases and mRNA capping enzymes (Shuman, S. and Schwer, B., Mol. Microbiol., 17:405-410 (1995); Timson, D. J., et al., Mut. Res., 460:301-318 (2000)). Until recently, the only clearly identifiable relatives of T4 RNA ligase, found through sequence comparisons (ex. with BLAST software), were from bacteriophage RB69 and Autographa californica nuclearpolyhedrosis virus. As disclosed in a previous patent applications (U.S. patent application Ser. No. 09/585,858; PCT Application No. PCT/1800/00893; European Application No. 00938977.6), the discovery of a bacteriophage from the thermophilic bacterial host Rhodothernus marinus and the subsequent genome sequencing identified a potential new RNA ligase belonging to this family according to the amino acid sequence of the predicted gene product of a particular open reading frame.
The use of thermostable enzymes has revolutionized the field of recombinant DNA technology. Thermostable enzymes, foremost DNA polymerases used in amplification of DNA, are of great importance in the research industry today. In addition, thermophilic enzymes are also used in commercial settings (e.g., proteases and lipases used in washing powder, hydrolytic enzymes used in bleaching). Identification of new thermophilic enzymes will facilitate continued DNA research as well as assist in improving commercial enzyme-based products.